喉鳞癌患者树突状细胞（DCs）功能及Hep-2总RNA转染DCs抗喉癌的实验研究

发布时间：2018-05-19 06:44

本文选题：树突状细胞 + 流式细胞仪　；参考：《山东大学》2008年博士论文

【摘要】： 【研究背景与目的】喉癌是耳鼻喉科常见的恶性肿瘤之一,发病率处于耳鼻咽喉各部分恶性肿瘤的第三位。在喉癌的病理类型中,以喉鳞癌最为常见,一般占全部喉恶性肿瘤的95～99%。目前喉癌的治疗以手术、放疗及化疗为主。近年来,虽然手术、放化疗技术发展迅速,但喉鳞癌患者特别是晚期患者的的长期生存率在过去10年中未见明显提高。因此,进一步改善喉癌患者预后的研究势在必行。以往的研究证实,抗肿瘤免疫在肿瘤发生发展的过程中有重要作用。机体的抗肿瘤免疫应答是以T淋巴细胞、NK细胞和巨噬细胞介导的细胞免疫应答为主。在此过程中,首先由抗原递呈细胞(antigen-presenting cell,APC)捕获、加工处理抗原并将抗原信息传递给T淋巴细胞,才能进而引发特异性免疫应答。树突状细胞(dendritic cell,DC)是已知体内功能最强的APC,能在体内外直接激活初始T细胞(na(?)ve T cell),促进辅助性T细胞和细胞毒性T细胞的生成,同时也能够促进B淋巴细胞生成抗体,是启动、调控并维持免疫应答的中心环节。由于DC在体内免疫应答中重要而独特的作用,近年来,以DC为基础的肿瘤免疫治疗研究越来越受到关注。已有研究证实,通过体外细胞因子联合培养扩增DC,模拟DC体内成熟过程,以不同形式的抗原物质,如人工合成肿瘤多肽、肿瘤细胞裂解产物、肿瘤蛋白抗原、凋亡肿瘤细胞、DNA或RNA负载DC,再将致敏DC回输体内作为疫苗可诱导机体产生有效的抗肿瘤免疫应答。树突状细胞应用于抗喉鳞癌的研究尚处于初级阶段。由于目前尚未发现喉癌肿瘤特异性抗原,因此肿瘤全抗原成为制备疫苗的主要手段。已有报道应用喉癌细胞裂解产物负载树突状细胞或用电融合的方法制备疫苗诱导产生有效的抗肿瘤免疫反应。最近研究发现,以RNA负载DC能够引发更为强大的抗肿瘤效应,而且已有实验证实了其优越的安全性及广阔的应用前景。但以RNA负载DC的抗喉鳞癌研究国内外尚未见报道。另外,树突状细胞免疫学功能的强弱取决于DC的免疫状态。众所周知,肿瘤患者的DC存在缺陷,但由于T细胞活化的MHC限制性,临床上应用DC诱导抗肿瘤免疫应答必须使用肿瘤患者自身的DC,因而研究恶性肿瘤患者DC的数目及功能显得尤为重要。另一方面,虽然DC为基础的免疫治疗有其独特的优势,但近期内仍无法作为主要的临床治疗手段。对于喉鳞癌患者,手术及放疗仍然不失为目前最有效的治疗方法。因此研究喉鳞癌患者DC免疫状态,除了考虑疾病本身对DC的影响,尚需考虑目前常规治疗方法对DC的影响。本研究检测手术及术后辅助放疗对喉癌患者外周血循环DC亚型及外周血单核细胞来源DC(monocyte-derived DCs,MoDes)功能的影响,以探讨喉癌免疫治疗的时机;同时,以EGFP标记的喉癌Hep-2株总RNA转染MoDCs,研究其诱导Hep-2细胞特异性的细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)的能力及对喉鳞癌的特异性杀伤效应,为临床以DC为基础的抗喉癌治疗提供实验基础和理论依据。第一部分健康人外周血树突状细胞亚型检测及培养、鉴定【目的】建立简便可靠的外周血树突状细胞亚型检测方法,培养鉴定外周血单核细胞来源树突状细胞。【方法】采集15例健康人外周血,采用三色流式细胞计量术检测外周血HLA-DR~+lineage~-树突状细胞及其亚型myeloid DC(mDC)、plasmacytoid DC(pDC)在外周血WBC所占百分比并计算其绝对数。采用淋巴细胞分离液自外周血分离获得单核细胞,体外经人重组细胞因子GM-CSF、IL-4和TNF-α诱导培养,获得成熟外周血单核细胞来源DC。倒置显微镜下观察DC形态,透射电镜观察DC超微结构,流式细胞仪检测其表型,混合淋巴细胞反应(MLR)检测刺激自体T淋巴细胞增殖的能力。【结果】健康人LIN~-DR~+细胞在WBC中所占百分比为0.41±0.13%,绝对数为22.75±5.22/μl,mDC在WBC中所占百分比为0.28±0.13%,绝对数为15.12±4.66/μl,pDC在WBC中所占百分比为0.068±0.032%,绝对数为3.78±1.40/μl;培养获得具有典型形态特征的外周血单核细胞来源DC;健康人LIN阴性细胞中,共刺激分子CD80(B7-1)、CD86(B7-2)阳性细胞所占百分比分别为46.5±7.1%和51.1±9.6%,同时多数DC表达DC成熟标志CD83和HLA-DR,阳性细胞百分比分别为62.6±10.3%和73.4±8.1%,表明单核细胞经细胞因子诱导后大部分分化为成熟DC;证实少量DC即可刺激自体淋巴细胞产生较强的增殖效应。【结论】流式细胞仪检测外周血DC亚群简便、可靠;联合应用细胞因子GM-CSF、IL-4和TNF-α能够自外周血中成功分离、诱导培养具有典型形态特征及功能活性的单核细胞来源DC。第二部分手术及术后放疗对喉鳞癌患者外周血树突状细胞亚型及功能的影响【目的】研究手术及术后辅助放疗对喉鳞癌患者外周血树突状细胞亚型及外周血单核细胞来源树突状细胞功能的影响。【方法】采集46例经病理证实的喉鳞癌患者及15例健康志愿者外周血。46名喉鳞癌患者中男性44例,女性2例,年龄分布为32～76岁,平均年龄为57.57±10.09岁。根据2002年国际抗癌联盟(UICC)制定的TNM分期标准,Ⅰ期14例,Ⅱ期8例,Ⅲ期11例,Ⅳ期13例。46例患者入院后均接受手术治疗,28例患者手术后接受颈部直线加速器放射治疗。接受单纯手术治疗的患者(n=18)及接受手术+放疗的患者(n=28)均于治疗前、治疗中及治疗后分三次采集外周血标本。采用三色流式细胞计量术检测外周血HLA-DR~+lineage~-树突状细胞及其亚型mDC、pDC在外周血WBC所占百分比并计算其绝对数。采用淋巴细胞分离液自外周血分离获得单核细胞,体外经人重组细胞因子GM-CSF、IL-4和TNF-α诱导培养,获得外周血单核细胞来源DC,流式细胞仪检测其表型,混合淋巴细胞反应(MLR)检测刺激自体T淋巴细胞增殖的能力。【结果】治疗前喉鳞癌患者外周血mDC计数、MoDC表面分子表达及刺激自体T淋巴细胞增殖能力均低于健康人;经手术治疗后,接受辅助放疗和未接受辅助放疗的患者其mDC计数、MoDC表面分子CD80,CD83,HLA-DR的表达均明显高于治疗前,但仅在术后未接受辅助放疗的患者中观察到CD86表达及刺激自体T淋巴细胞增殖能力的恢复。【结论】喉鳞癌患者DC存在缺陷;手术治疗可提高喉鳞癌患者外周血mDC计数及MoDC的功能;术后辅助放疗不影响外周血mDC计数的正常化,但对MoDC功能具有抑制作用。第三部分以EGFP标记的喉癌Hep-2细胞株总RNA转染树突状细胞的特异性抗喉癌实验研究【目的】探讨以EGFP为标记观察喉鳞癌Hep-2细胞株总RNA转染树突状细胞的可行性;探讨转染后DC诱导特异性识别Hep-2细胞的细胞毒性T淋巴细胞的可能性及特异性杀伤效应。【方法】采用淋巴细胞分离液自健康人外周血分离获得单核细胞,体外经人重组细胞因子GM-CSF、IL-4诱导培养,获得未成熟DC,观察细胞形态,流式细胞技术鉴定其表型;培养喉癌Hep-2细胞株,采用脂质体转染技术将携带绿色荧光蛋白质粒pEGFP-N1转染至Hep-2细胞株,通过G418筛选稳定表达EGFP的细胞株;Trizol一步法提取稳定转染的Hep-2细胞株总RNA,检测其完整性及纯度;以共孵育及电穿孔法将RNA导入未成熟DC,并以TNF-α促成熟;应用荧光显微镜观察并计算转染效率;通过流式细胞技术、混合淋巴细胞反应检测RNA转染后对DC表型、刺激T细胞增殖能力的影响;将转染后DC与同种异体T淋巴细胞共培养7天,收集效应细胞作为特异性CTL,通过~(51)Cr释放法检测其对Hep-2细胞株的特异性杀伤效应。【结果】pEGFP-N1质粒转染喉癌Hep-2细胞株,经G418筛选可获得稳定表达EGFP的细胞株,荧光显微镜下可见绿色荧光,经20次传代后荧光仍无消失,Hep-2细胞形态及生长特性未见明显改变;经Trizol一步法提取稳定转染的Hep-2细胞株总RNA 80～100μg/10~7细胞,分光光度法测定OD260/OD280=1.83,RNA电泳显示18S、28S条带,证实RNA的完整性;共孵育及电穿孔转染法转染效率分别为7～9%和21～23%;RNA导入未成熟DC后,细胞表达绿色荧光持续20～24小时;Hep-2-EGFP-RNA转染组DC表面分子CD83、HLA-DR表达较转染前及对照转染组显著升高,阳性细胞百分比达89.5%、95.4%,刺激同种异体T淋巴细胞增殖的能力显著提高;Hep-2-EGFP-RNA转染组DC体外诱导的CTLs可特异性杀伤喉癌Hep-2细胞,但对结肠癌HT-29细胞及卵巢癌OVCAR3细胞无杀伤活性。【结论】绿色荧光蛋白可作为喉癌Hep-2细胞RNA转染DC的标记;转染喉癌Hep-2细胞总RNA促进DC成熟,提高DC刺激同种异体T淋巴细胞增殖的能力,并能够产生喉癌Hep-2细胞特异性杀伤效应。
[Abstract]:Background and purpose of research

In recent years , the long - term survival rate of laryngeal squamous cell carcinoma , especially advanced patients , has not been significantly improved in the past 10 years . Therefore , it is imperative to further improve the prognosis of laryngeal cancer patients .

Dendritic cells ( DCs ) are the most powerful APC in vivo , which can promote the production of T - cells and cytotoxic T - cells , but also promote the production of antibody against B - lymphocytes , which is the central link of starting , regulating and maintaining immune response .

Dendritic cells ( DCs ) have been used in the study of anti - laryngeal squamous cell carcinoma ( SCC ) .

It is well known that DC - induced antitumor immune responses in patients with laryngeal squamous cell carcinoma have a unique advantage . However , although DC - based immunotherapy has its unique advantages , the DC - induced immune response in patients with laryngeal squamous cell carcinoma is not currently the most effective treatment . In addition to the effect of the disease itself on DC , the effect of conventional treatment methods on DC is still needed .

The effects of surgical and postoperative adjuvant radiotherapy on peripheral circulating DC ( DC ) and peripheral blood mononuclear cells ( DCs ) in peripheral blood mononuclear cells ( DCs ) of patients with laryngeal cancer were investigated . The cytotoxic T lymphocyte ( CTL ) and specific killing effects on laryngeal squamous cell carcinoma ( SCC ) were investigated by using EGFP - labeled total RNA of laryngeal cancer .

Detection , Culture and Identification of Dendritic Cell Subtypes in Human Peripheral Blood in the First Part

Objective To establish a simple and reliable method for detecting peripheral blood mononuclear cells ( DCs ) from peripheral blood mononuclear cells ( PBMC ) .

Peripheral blood mononuclear cells ( HLA - DR ~ + ) ~ - dendritic cells and their subtypes , DC ( mDC ) and plasmacytoid DC ( pDC ) were measured by three - color flow cytometry .

The percentage of DCs in WBC was 0 . 28 卤 0 . 13 % , the absolute number was 22.75 卤 5.22 / 渭l , the percentage of mDC in WBC was 0 . 068 卤 0 . 32 % , the absolute number was 3.78 卤 1.40 / 渭l , the percentage of positive cells was 62.6 卤 10.3 % and 73.4 卤 8.1 % , respectively .

Conclusion Flow cytometry is simple and reliable in detecting peripheral blood DC subgroup , and the combination of cytokine GM - CSF , IL - 4 and TNF - 伪 can be successfully isolated from peripheral blood , which can induce DC of monocytes with typical morphological characteristics and functional activity .

Effects of second partial surgery and postoperative radiotherapy on the subtypes and functions of peripheral blood dendritic cells in patients with laryngeal squamous cell carcinoma

Objective To investigate the effects of surgical and postoperative adjuvant radiotherapy on the function of dendritic cells in peripheral blood dendritic cells and peripheral blood mononuclear cells in patients with laryngeal squamous cell carcinoma .

Forty - six patients with laryngeal squamous cell carcinoma and 15 healthy volunteers were collected from peripheral blood of 46 patients with laryngeal squamous cell carcinoma .

The expression of CD80 , CD83 and HLA - DR in peripheral blood of patients with laryngeal squamous cell carcinoma was significantly higher than that before treatment . The expression of CD80 , CD83 and HLA - DR on the surface of MoDC was significantly higher than that before treatment , but only in patients who did not receive adjuvant radiotherapy after surgery .

Conclusion There is a deficiency in DC in patients with laryngeal squamous cell carcinoma . Surgical treatment can improve the peripheral blood mDC count and MoDC function of patients with laryngeal squamous cell carcinoma . The postoperative adjuvant radiotherapy does not affect the normalization of mDC count in peripheral blood , but has an inhibitory effect on MoDC function .

Experimental study on the specific anti - laryngocarcinoma of dendritic cells transfected with EGFP - labeled total RNA of laryngeal cancer cells

Objective : To investigate the feasibility of using EGFP as a marker to observe the expression of dendritic cells transfected with total RNA from laryngeal squamous cell carcinoma cells .

Cells were isolated from the peripheral blood of healthy individuals using lymphocyte separation liquid . The cells were cultured in vitro by human recombinant cytokines GM - CSF and IL - 4 . The cell lines were transfected with green fluorescent protein particles by G418 selection , the integrity and purity of the cells were observed . The transfection efficiency was observed by G418 selection . After transfection , the cells were cultured for 7 days . The effect cells were collected as specific CTL .

Results The expression of CD83 and HLA - DR was significantly higher than that in control transfected group . The expression of CD83 , HLA - DR was 89.5 % and 95.4 % , respectively , and the expression of CD83 and HLA - DR was 89.5 % and 95.4 % respectively .

Conclusion The green fluorescent protein can be used as a marker for the transfection of DC by RNA transfection of laryngeal cancer 2 - cell RNA . The transfection of total RNA of laryngeal cancer 2 - 2 cells promotes DC maturation , enhances the ability of DC to stimulate the proliferation of allogeneic T lymphocytes , and can produce the specific killing effect of laryngeal cancer Hep2 cells .
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392;R739.65

【引证文献】